After more than 30 years of using ?-lactamase inhibitors (BLIs) in the treatment of patients, pressing questions still remain at the forefront of our clinical efforts. Although, the value of ?-lactam/BLI combinations (e.g., piperacillin/tazobactam) was well-established by the 1990s, the rapid emergence of ?-lactamases that hydrolyzed expanded spectrum cephalosporins, carbapenems and were resistant to inactivation by BLIs created a global crisis in antimicrobial chemotherapy and propelled the quest for a novel class of inhibitors, the diazabicyclooctanes (DBOs). In addition to the extended spectrum ?-lactamases (ESBLs), the major challenges to overcome in Klebsiella pneumoniae are the serine carbapenemases (e.g., KPC-2, OXA-48) and the metallo- ?-lactamases (MBLs) (e.g., NDM-1, VIM and IMP). Fortunately, avibactam (AVI), a DBO BLI, inactivates KPC-2 and OXA-48; the efficacy of AVI against MBLs is absent. Our investigations in the previous grant cycle awarded us with unprecedented and frightening insights. We were painfully reminded that we cannot anticipate ?-lactamase evolution; the diversity in amino acid sequences that nature can yield and their impact on catalytic activity and resistance are unpredictable. We found to our dismay that KPC-2 ?-lactamase variants expressed in Escherichia coli DH10B with the amino acid substitutions, S130G, K234R, and R220M conferred resistance to ampicillin-AVI. Moreover, we discovered that a single amino acid substitution in VIM-24 (R228L) confers enhanced resistance to ceftazidime and cefepime. We came to the inevitable conclusion that: i) resistance to AVI was present before this BLI was released; and ii) MBLs can expand their substrate profile and enhance cephalosporin resistance much like the class A and C extended-spectrum ?-lactamases (ESBLs). In this Merit application, our goals are to: 1) continue to probe the structural and mechanistic basis for resistance to AVI in KPC; 2) learn how novel substrate specificity evolves in KPC and NDM carbapenemases. We will apply for the first time complementary structural methods (x-ray crystallography, NMR, and Double Electron Electron Resonance, DEER) to help us understand how structure activity relationship are defined in carbapenemases with the intent that these new insights will lead to new approaches in BLI design. In addition, recent computational analyses of KPC-2 ?-lactamase molecular dynamics predict that ?hydrophobic networks? contribute to the structural integrity and allosteric signaling of KPC-2 and other class A ?-lactamases. This novel insight defines our third goal: 3) if these hydrophobic networks contribute to allosteric signaling, can allosteric inhibitors offer new opportunities for BLI design. The unrelenting pace of resistance makes it imperative that we understand fundamental biochemical interactions in order to find BLIs that act by novel mechanisms. Our multidisciplinary approach to studying carbapenemases will allow us to anticipate how new mutations will effect BLI and cephem catalysis and anticipate novel resistance phenotypes. We also propose that we will discover important conformational changes upon AVI binding and uncover networks that may be involved in allosteric signaling. If this is confirmed, disruption of these ?hydrophobic networks? will open an alternative approach to overcoming resistance and lead to new BLIs.